First Dimension Separation Research Articles

Trimodal Mixed Mode Chromatography That Enables Efficient Offline Two-Dimensional Peptide Fractionation for Proteome Analysis.

Offline two-dimensional chromatography is a common means to achieve deep proteome coverage. To reduce sample complexity and dynamic range and to utilize mass spectrometer (MS) time efficiently, high chromatographic resolution of and good orthogonality between the two dimensions are needed. Ion exchange and high pH reversed phase chromatography are often used for this purpose. However, the former requires desalting to be MS-compatible, and the latter requires fraction pooling to create orthogonality. Here, we report an alternative first-dimension separation technique using a commercial trimodal phase incorporating polar embedded reversed phase, weak anion exchange, and strong cation exchange material. The column is capable of retaining polar and nonpolar peptides alike without noticeable breakthrough. It allows separating ordinary and TMT-labeled peptides under mild acidic conditions using an acetonitrile gradient. The direct MS compatibility of solvents and good orthogonality to online coupled C18 columns enable a straightforward workflow without fraction pooling and desalting while showing comparable performance to the other techniques. The method scales from low to high microgram sample quantity and is amenable to full automation. To demonstrate practical utility, we analyzed the proteomes of 10 human pancreatic cancer cell lines to a depth of >8,700 quantified proteins.

Exploratory Purification of Compounds from Aqueous Extracts of Pinus massoniana Lamb. by Two-Dimensional Preparative Liquid Chromatography

An off-line orthogonal two-dimensional preparative liquid chromatography method was developed for isolation of high-purity compounds from aqueous extracts of Pinus massoniana Lamb., which has efficient therapeutic properties in recipes of traditional Chinese medicines. A polar-enhanced reversed-phase (RP) column, XAqua C18 was selected for the first-dimensional separation. A HILIC column and a hydrophobic RP column were both explored in the second dimension. The preparation results indicated hydrophilic RP × HILIC mode would provide better separation efficiency for polar complexes, i.e., the second-dimensional separation was finished in less than 30 min and high-purity compounds were obtained. In this work, 16 fractions were collected efficiently in the first dimension with a recovery up to 96%, and six compounds with high purity and enough quantity were identified by NMR and high resolution mass spectrometry, including epicatechin, betuloside, taxifolin-3-O-xyloside, cedrusin-4-O-glucoside, massonianoside C and massonianoside D, and five are glycosides, including flavonoid glycosides, lignanoid glycosides and an arylbutanoid glycoside. Betuloside was first reported in the preparation of Pinus massoniana Lamb. This work demonstrated a flexible strategy of compromise in between time and resolution for selecting solvent and column to enhance preparative efficiency of polar complex systems.

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

Development of a volatility and polarity separator (VAPS) for volatility- and polarity-resolved organic aerosol measurement

Discrepancies between modeled and measured atmospheric organic aerosol (OA) have highlighted the need for in situ instrumentation to better characterize the sources, formation mechanisms, and atmospheric evolution of ambient OA. We have developed the Volatility and Polarity Separator (VAPS) for hourly measurements of volatility- and polarity-resolved OA detected using high-resolution time-of-flight mass spectrometry (HR-ToF-MS). Here, atmospheric OA is inertially impacted onto a collection cell, material is transferred onto a short transfer line located inside a gas chromatography (GC) oven, the oven is heated to provide a first-dimension separation of volatility, then thermally pulsed through a short polar GC column for a second-dimension polarity separation, and finally detected by HR-ToF-MS. This novel instrument increases the mass throughput of ambient OA in comparison to traditional GC due to shorter transfer paths and passivated coatings. Molecular separation resolution is partially sacrificed for this increased mass recovery, but the high-resolution mass spectral data recovers information such as chemical classes and even some individual compounds along with elemental composition to determine aerosol oxidation states. Different techniques for interpreting and representing VAPS data are considered and its applicability to positive matrix factorization (PMF) analysis is demonstrated.Copyright © 2016 American Association for Aerosol Research

Design and evaluation of microfluidic devices for two-dimensional spatial separations

Various designs of chips for comprehensive two-dimensional spatial liquid chromatography were investigated. The performance of these chips was initially evaluated using computational fluid dynamics (CFD). A bifurcating distributor with an angle of 140° between branches was implemented in order to achieve a homogeneous velocity field. The cross-sectional area of the channels of the flow distributor was fixed at 0.5×0.5mm, which allows a robust micromilling technique to be used for chip manufacturing. Experiments were performed with chips featuring purposely introduced imperfections in the structure of the bifurcating flow distributor to study its capacity of overcoming potential local clogging. Split peaks were observed when 75% of one of the flow channels was obstructed, in line with the CFD predictions. The main bottlenecks for the performance of the spatial two-dimensional chips were identified, viz. sample injected in the first dimension diverging into the flow distributor and channel discretization (i.e., remixing of first-dimension separation peaks because of finite number of second-dimension channels). Solutions to the former problem were studied by applying a flow resistance in the vertical segments that formed the outlets of the flow distributor and by simulating the presence of constrictions. It was found that a flow resistance of 1.0×1011m−2 reduced the amount of sample diverging into the flow distributor by a factor of 10. The presence of a constriction of 90% of the segment area and 50% of the segment length decreased the diverging flow by a factor of 5. The influence of the linear velocity was significant. Solutions to the channel discretization problem were sought by investigating different designs of spatial two-dimensional chips.

Analyses of Total Alkaloid Extract of Corydalis yanhusuo by Comprehensive RP × RP Liquid Chromatography with pH Difference.

A comprehensive two-dimensional (2D) reverse phase (RP) liquid chromatography (LC) method is developed for alkaloid analysis. This offline comprehensive 2D method is developed using different pH values. With a pH value of 10.5, most alkaloids appear in the form of neutral molecules possessing high retention factors based on their polarity, while the alkaloid polarity order is changed when the pH value decreased to 3.0. The performance of pH modulated 2D LC is demonstrated with 8 alkaloid standards which resulted in orthogonal separation. The developed method is then applied to total alkaloid separation in Corydalis yanhusuo. The first-dimension separation is carried out using methanol and water containing 1.0% ammonium hydroxide and a strong base-resistant RP column, which afforded a peak capacity of 94. The second-dimension analysis is carried out with a surface positive charge column providing a peak capacity of 205 using a mobile phase consisting of acetonitrile and water with 0.15% formic acid. 2D analyses of total alkaloid extract from C. yanhusuo afford a total peak capacity of 9090. Sixteen compounds were tentatively identified based on their ultraviolet spectrum and MS/MS analyses. The proposed method provides an alternative approach to achieve high peak capacity for analysis of alkaloid extract.

Online comprehensive two-dimensional ion chromatography × capillary electrophoresis.

A comprehensively coupled online two-dimensional ion chromatography-capillary electrophoresis (IC × CE) system for quantitative analysis of inorganic anions and organic acids in water is introduced. The system employs an in-house built sequential injection-capillary electrophoresis instrument and a nonfocusing modulation interface comprising a tee-piece and a six-port two-position injection valve that allows comprehensive sampling of the IC effluent. High field strength (+2 kV/cm) enables rapid second-dimension separations in which each peak eluted from the first-dimension separation column is analyzed at least three times in the second dimension. The IC × CE approach has been successfully used to resolve a suite of haloacetic acids, dalapon, and common inorganic anions. Two-dimensional peak capacity for IC × CE was 498 with a peak production rate of 9 peaks/min. Linear calibration curves were obtained for all analytes from 5 to 225 ng/mL (except dibromoacetic acid (10-225 ng/mL) and tribromoacetic acid (25-225 ng/mL)). The developed approach was used to analyze a spiked tap water sample, with good measured recoveries (69-119%).

Design of a microfluidic device for comprehensive spatial two-dimensional liquid chromatography.

This study discusses the design aspects for the construction of a microfluidic device for comprehensive spatial two-dimensional liquid chromatography. In spatial two-dimensional liquid chromatography each peak is characterized by its coordinates in the plane. After completing the first-dimension separation all fractions are analyzed in parallel second-dimension separations. Hence, spatial two-dimensional liquid chromatography potentially provides much higher peak-production rates than a coupled column multi-dimensional liquid chromatography approach in which the second-dimension analyses are performed sequentially. A chip for spatial two-dimensional liquid chromatography has been manufactured from cyclic olefin copolymer and features a first-dimension separation channel and 21 parallel second-dimension separation channels oriented perpendicularly to the former. Compartmentalization of first- and second-dimension developments by physical barriers allowed for a preferential flow path with a minimal dispersion into the second-dimension separation channels. To generate a homogenous flow across all the parallel second-dimension channels, a radially interconnected flow distributor containing two zones of diamond-shaped pillars was integrated on-chip. A methacrylate ester based monolithic stationary phase with optimized macroporous structure was created in situ in the confines of the microfluidic chip. In addition, the use of a photomask was explored to localize monolith formation in the parallel second-dimension channels. Finally, to connect the spatial chip to the liquid chromatography instrument, connector ports were integrated allowing the use of Viper fittings. As an alternative, a chip holder with adjustable clasp locks was designed that allows the clamping force to be adjusted.

Continuous vs. segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.).

A comprehensive two-dimensional liquid chromatography system in combination with photodiode array and mass spectrometry detection was developed for analysis of polyphenols in sugarcane (Saccharum spp.) leaf extracts. To achieve this, a micro cyano column and a partially porous octodecylsilica column were used in the first and the second dimension, respectively. The choice of the cyano column over other reversed-phase columns tested for the first-dimension separation was due to its lower correlation selectivity with respect to the octodecylsilica column, which was used for the second-dimension separation. Even when reversed-phase mode was used in both dimensions, a satisfactory degree of orthogonality was achieved by use of different gradient elution modes in the second dimension. By means of the setup investigated, 38 polyphenolic compounds were detected, and among them 24 were positively identified by means of complementary data from photodiode array and mass spectrometry detection and an in-house database. This is the first time such a powerful analytical technique has been used for polyphenolic characterization of sugarcane extracts.

Membrane-assisted isoelectric focusing device as a micropreparative fractionator for two-dimensional shotgun proteomics.

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.

The One Hour Yeast Proteome

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

Separation of Fatty Acid Methyl Esters by GC-Online Hydrogenation × GC

The separation of fatty acid methyl esters (FAME) provided by a 200 m × 0.25 mm SLB-IL111 capillary column is enhanced by adding a second dimension of separation ((2)D) in a GC × GC design. Rather than employing two GC columns of different polarities or using different elution temperatures, the separation in the two-dimensional space is achieved by altering the chemical structure of selected analytes between the two dimensions of separation. A capillary tube coated with palladium is added between the first dimension of separation ((1)D) column and the cryogenic modulator, providing the reduction of unsaturated FAMEs to their fully saturated forms. The (2)D separation is achieved using a 2.5 m × 0.10 mm SLB-IL111 capillary column and separates FAMEs based solely on their carbon skeleton. The two-dimensional separation can be easily interpreted based on the principle that all the saturated FAMEs lie on a straight diagonal line bisecting the separation plane, while the FAMEs with the same carbon skeleton but differing in the number, geometric configuration or position of double bonds lie on lines parallel to the (1)D time axis. This technique allows the separation of trans fatty acids (FAs) and polyunsaturated FAs (PUFAs) in a single experiment and eliminates the overlap between PUFAs with different chain lengths. To our knowledge, this the first example of GC × GC in which a chemical change is instituted between the two dimensions to alter the relative retentions of components and identify unsaturated FAMEs.

Elucidation of steroid glycosides in Anemarrhena asphodeloides extract by means of comprehensive two‐dimensional reversed‐phase/polyamine chromatography with mass spectrometric detection

In the work presented here, a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Reversed-phase liquid chromatography was employed for the first-dimension separation and polyamine chromatography was chosen for the second-dimension separation mode. The two dimensions are highly orthogonal and the separation efficacy of the developed octadecylsilica × polyamine was tested by separating an extract from Anemarrhena asphodeloides. The steroid glycosides identified by comprehensive two-dimensional liquid chromatography in this experiment were compared to those obtained for monodimensional liquid chromatography. The comprehensive two-dimensional liquid chromatography system, thanks to the complementary separation selectivity and enhanced peak capacity provided by the two columns, allowed to distribute five compounds of low amounts otherwise unachievable by monodimensional liquid chromatography. In addition, four steroid isomers with similar fragmentation characteristics in MS/MS spectra, were newly separated based on their different chemical structures.

Separation of Polypeptides by Isoelectric Point Focusing in Electrospray-Friendly Solution Using a Multiple-Junction Capillary Fractionator

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.

Two multidimensional chromatographic methods for enantiomeric analysis of o,p′-DDT and o,p′-DDD in contaminated soil and air in a malaria area of South Africa

In rural parts of South Africa the organochlorine insecticide DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) is still used for malaria vector control where traditional dwellings are sprayed on the inside with small quantities of technical DDT. Since o,p′-DDT may show enantioselective oestrogenicity and biodegradability, it is important to analyse enantiomers of o,p′-DDT and its chiral degradation product, o,p′-DDD, for both health and environmental-forensic considerations. Generally, chiral analysis is performed using heart-cut multidimensional gas chromatography (MDGC) and, more recently, comprehensive two-dimensional gas chromatography (GC×GC). We developed an off-line gas chromatographic fraction collection (heart-cut) procedure for the selective capturing of the appropriate isomers from a first apolar column, followed by reinjection and separation on a second chiral column. Only the o,p′-isomers of DDT and DDD fractions from the first dimension complex chromatogram (achiral apolar GC column separation) were selectively collected onto a polydimethylsiloxane (PDMS) multichannel open tubular silicone rubber trap by simply placing the latter device on the flame tip of an inactivated flame ionisation detector (FID). The multichannel trap containing the o,p′-heart-cuts was then thermally desorbed into a GC with time-of-flight mass spectrometry detection (GC–TOFMS) for second dimension enantioselective separation on a chiral column (β-cyclodextrin-based). By selectively capturing only the o,p′-isomers from the complex sample chromatogram, 1D separation of ultra-trace level enantiomers could be achieved on the second chiral column without matrix interference. Here, we present solventless concentration techniques for extraction of DDT from contaminated soil and air, and report enantiomeric fraction (EF) values of o,p′-DDT and o,p′-DDD obtained by a new multidimensional approach for heart-cut gas chromatographic fraction collection for off-line second dimension enantiomeric separation by 1D GC–TOFMS of selected isomers. This multidimensional method is compared to the complementary technique of comprehensive GC×GC–TOFMS using the same enantioselective column, this time as the first dimension of separation.

Application of the ProteomeLab™ PF2D protein fractionation system in proteomic analysis for human genetic diseases

Abstract Proteomic analysis has been widely used in elucidating the mechanism of diseases. As a classical proteomic approach, two-dimensional gel electrophoresis (2DGE) has been commonly applied in finding differentially expressed proteins through a first dimension of separation by the isoelectric point (pI) of proteins and a second dimension of separation according to the molecular weight (MW) of proteins. Compared to 2DGE, a recently developed commercial system from Beckman Coulter, the two-dimensional protein fractionation (PF2D), separates proteins according to the pI of proteins in the first dimension followed by a second dimension of separation according to the degree of protein hydrophobicity. As a liquid-based fractionation system, PF2D could facilitate the extraction and separation of broader protein categories and improve reproducibility and quantification as well as be less labor-intensive, which are usually identified as limitations of a gel-based 2DGE platform. This review evaluates the applications of the PF2D system and discusses the perspectives and advantages of PF2D in the investigation of cancer and genetic disorders and in protein mapping in human biological fluids and cell cultures.

Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography and Isoelectric Focusing Techniques

Abstract Efficient pre-purification steps for the enrichment of phosphorylated proteins or phosphopeptides are necessary for the detection of phosphorylation sites in phosphoproteomic analysis. Currently, the most common first-dimensional separation technique is strong cation exchange (SCX). A potential alternative to SCX-based separation is to use isoelectric focusing (IEF) as a first-dimensional separation technique, which has been demonstrated recently. In this article, we present a direct comparison between SCX and IEF based on IPG strips (IPG-IEF) for phosphoproteomic separation. The comparison experiments were carried out using standard phosphoproteins and a real sample of HepG2 cell. Then, the comparison of 18 O labeling phosphopeptides' stability under IPG-IEF with that under SCX was made. Fractions obtained from both the techniques (SCX and IPG-IEF) were analyzed using High Mass Accuracy LTQ-FTICR-MS/MS. The results demonstrated that SCX and IPG-IEF are useful in phosphopeptides enrichment analyses on a large scale and that SCX is superior to IPG-IEF, whereas the 18 O labeling phosphopeptides' stability under SCX is poor compared with that under IPG-IEF.

Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography-Isoelectric Focusing Techniques

Efficient pre-purification steps for the enrichment of phosphorylated proteins or phosphopeptides are necessary for better detection of phosphorylation sites in phosphoproteomic analysis.Currently,the most common first-dimensional separation technique used is strong cation exchange(SCX).A potential alternative to SCX-based separation is to use isoelectric focusing(IEF) as a first-dimensional separation technique,which has been demonstrated recently.In this study,we present a direct comparison between SCX and IEF based on IPG strips(IPG-IEF) for the phosphoproteomic separation.The comparison experiments discussed in this study utilized standard phosphoproteins and a real sample of HepG2 cell.Then the comparison of 18O labeling phosphopeptides′ stability under immobilized pH gradient gel(IPG)-IEF with under SCX was made.Fractions from both technique(SCX and IPG-IEF) were analyzed using the High Mass Accuracy LTQ-FTICR-MS/MS.The results demonstrate that SCX-LTQ-FT and IPG-IEF-LTQ-FT are useful in the phosphopeptides enrichment analysis on a large scale.And SCX-LTQ-FT is relative superior to IPG-IEF-LTQ-FT,whereas the 18O labeling phosphopeptides′stability under SCX-LTQ-FT is relatively poor with that under IPG-IEF-LTQ-FT.

Modification of Histones by Sugar β-N-Acetylglucosamine (GlcNAc) Occurs on Multiple Residues, Including Histone H3 Serine 10, and Is Cell Cycle-regulated

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.